[24] ARYL SULFOTRANSFERASES 197

PURIFICATION AND RECOVERY OF BILIRUBIN MONOGLUCURONIDEDISMUTASE ACTIVITY

FROM RAT LIVER

Bilirubin Specific
Total protein Volume diglucuronide activity
(rag) (ml) (nmol/min) (units/rag protein)

Plasma membrane
fraction 42.3 28 1200 28.4
Sonication 19.0 5 356 18.7
Agarose column 0.96 5 317 330
Isoelectric
focusing 0.081 8 178 2200

electrofocusing equipment using a 50 to 0% sucrose gradient containing

1% carrier ampholytes with a pH range of 7 to 9. Fractions of 2 ml are
collected in ice-cooled tubes, and those with enzyme activity are pooled
and concentrated by negative pressure dialysis. The purification proce-
dure is summarized in the table.

Properties
The enzyme has an apparent molecular weight of 160,000 by agarose
gel chromatography, and a subunit molecular weight of 28,000 by SDS gel
electrophoresis. The isoelectric point is 7.9. The Km for bilirubin mono-
glucuronide is 33/zM.

[24] A r y l S u l f o t r a n s f e r a s e s
By RONALD D . SEKURA, MICHAEL W. D U F F E L , a n d WILLIAM B. JAKOBY

Phenol + adenosine 3'-phosphate 5'-phosphosulfate

aryl sulfate + adenosine 3',5'-bisphosphate

This group of enzymes (EC 2.8.2.1) is active with a wide variety of

phenolic compounds as substrates. ~-~ At least four homogeneous en-
zymes with this activity have been isolated from the liver of Sprague-
W. B. Jakoby, R. D. Sekura, E. S. Lyon, C. J. Marcus, and J.-L. Wang, in "Enzymatic
Basis of Detoxication" (W. B. Jakoby, ed.), Vol. 2, p. 199. Academic Press, New York,
1980.
2 A. B. Roy, Handb. Exp. Pharmacol. [N.S.] 28, Part 2, 536 (1971).
3 R. D. Sekura and W. B. Jakoby, J. Biol. Chem. 254, 5658 (1979).
4 R. D. Sekura and W. B. Jakoby, Arch. Biochem. Biophys.. in press (1981).
'~ R. D. Sekura, K. Sato, H. J. Cahnmann, J. Robbins, and W. B. Jakoby, Endocrinology
108, 454 (1981).

Copyright© 1981by AcademicPress, Inc.

METHODS IN ENZYMOLOGY.VOL.77 All rightsof reproductionin any formreserved.
ISBN 0-12-181977-9
198 ENZYME PREPARATIONS [24]

D a w l e y rats. 3,4 T h e a r y l s u l f o t r a n s f e r a s e s fall into t w o families a n d are

d e s i g n a t e d b y r o m a n n u m e r a l s in the o r d e r o f d e c r e a s i n g isoelectric p o i n t s
o f t h e p r o t e i n s . T h e first f a m i l y , c o n s i s t i n g o f aryl s u l f o t r a n s f e r a s e s I a n d
II, c o m p r i s e s e n z y m e s h a v i n g e n t i r e l y s i m i l a r specificity for p h e n o l s ; with
2 - n a p h t h o l t h e y h a v e a p H o p t i m u m at a b o u t p H 6.5. 3 A r y l sulfo-
t r a n s f e r a s e s I I I a n d I V also r e s e m b l e e a c h o t h e r in t h e i r specificity for
p h e n o l s b u t , in a d d i t i o n , act w i t h o r g a n i c h y d r o x y l a m i n e s a n d w i t h pep-
tides b e a r i n g N - t e r m i n a l t y r o s i n e r e s i d u e s , as sulfate a c c e p t o r s . 4 W i t h a
s i m p l e p h e n o l as a c c e p t o r , i.e., 2 - n a p h t h o l , this latter g r o u p o f e n z y m e s
has o p t i m a l a c t i v i t y at p H 5.5.

Assay M e t h o d s

Principle. T h e a s s a y 9 t a k e s advantage o f the p a r t i t i o n into a n organic

s o l v e n t o f the ion p a i r f o r m e d b e t w e e n c e r t a i n o r g a n i c sulfate m o n o e s t e r s
a n d m e t h y l e n e b l u e . 2 - N a p h t h o l a n d P A P S '° s e r v e as s u b s t r a t e s .

Reagents
Buffer: 0.5 M s o d i u m p h o s p h a t e at p H 7.4 for the a s s a y o f sulfo-
t r a n s f e r a s e s I a n d II, or 0 . 5 M s o d i u m a c e t a t e at p H 5.5 for the a s s a y
of sulfotransferases III and IV

The assay presented here, with 2-naphthol as sulfate acceptor, is effective in following the
course of purification but not for evaluating the kinetics of the reaction or with other
substrates. A chromatographic assay Tis more versatile and is particularly useful, because
of its low blank values, in determining kinetic constants with poor substrates. Sulfate
transfer to phenols is assayed in a total volume of 50/xl containing 0.25 M buffer at the
appropriate pH; 5 mM 2-mercaptoethanol; the phenol at a concentration of 0.5 mM; less
than 5% acetone that is added along with the phenol; and 0.1 mM [35S]PAPS (about 120
/~Ci per ~mol). The reaction is initiated by addition of enzyme and, after 10 min at 37°, is
terminated by addition of 13 /.d of 2 M acetic acid. An aliquot of 5 /.d of the reaction
mixture is applied to a thin-layer strip (1 × 10 cm) of cellulose on a plastic backing
(Eastman No. 6064).Once the samples are dry, the chromatographic strips are developed
with n-propanol : ammonia : water (6 : 3 : 1) at 4°. After the solvent has moved about 8 cm,
the strips are removed and the solvent front is marked. With most substrates, a 2-cm
section is cut below and including the solvent front and placed directly into a scintillation
vial containing 10 ml of Aquasol. With more polar substrates, such as those containingfree
amino groups, product must be localized by autoradiography.
With [35S]PAPSthat has been purified by chromatography on DEAE-cellulose, a there is
essentially no radioactive contaminant that migrates with the sulfate esters.
7 R. D. Sekura, C. J. Marcus, E. S. Lyon, and W. B. Jakoby, Anal. Biochem. 95, 83 (1979).
8 Commercially available 3~S-labeledPAPS contains material that migrates to the solvent
front when used in the chromatographic assay. ~ The radioactive PAPS can be readily
purified by applying 250/~Ci of the nucleotide in 50% ethanol to a column (0.9 × 10 era) of
DE-52 (Whatman) that has been equilibrated with 50 mM triethylamine-carbonate at pH
[24] ARYL SULFOTRANSFERASES 199

2 - M e r c a p t o e t h a n o l , 0.1 M , p r e p a r e d b y a d d i n g 0.07 m l o f t h e thiol to

I0 ml w a t e r
2 - N a p h t h o l , 5 m M , d i s s o l v e d in a c e t o n e 11
PAPS, 4 mM
M e t h y l e n e b l u e r e a g e n t : 250 m g m e t h y l e n e b l u e , 50 g a n h y d r o u s
s o d i u m sulfate, a n d 10 ml H2SO4 a r e d i s s o l v e d in w a t e r to a final
v o l u m e o f 1 liter
Procedure. T h e f o l l o w i n g a r e a d d e d s e q u e n t i a l l y , r e s u l t i n g in a t o t a l
v o l u m e o f 0.4 ml: w a t e r ; 0.2 ml b u f f e r solution; 3 0 / z l 2 - m e r c a p t o e t h a n o l ;
20/.d 2 - n a p h t h o l ; 20 ttl P A P S ; e n z y m e . 12 F o l l o w i n g t h e a d d i t i o n o f e n z y m e
a n d i n c u b a t i o n at 37 ° for 10 min, t h e r e a c t i o n is t e r m i n a t e d w i t h 0.5 ml o f
m e t h y l e n e b l u e r e a g e n t f o l l o w e d b y 2 ml o f c h l o r o f o r m . A f t e r a g i t a t i o n
w i t h a v o r t e x m i x e r for 20 sec, t h e s u s p e n s i o n is c e n t r i f u g e d at 2 0 0 0 g for
10 min. C h l o r o f o r m , t h e l o w e r l a y e r , is t r a n s f e r r e d to a t u b e c o n t a i n i n g
b e t w e e n 50 to 100 m g o f a n h y d r o u s s o d i u m sulfate. A b s o r b a n c e is d e t e r -
m i n e d a t 651 n m ; 10 n m o l o f e i t h e r 4 - n i t r o p h e n y l s u l f a t e o r 1 - n a p h t h y l
s u l f a t e , c a r r i e d t h r o u g h t h e e x t r a c t i o n p r o c e d u r e , y i e l d a n A651 o f 0.3.
Definition o f Unit. A unit o f e n z y m e is t h e a m o u n t n e c e s s a r y f o r the
c a t a l y s i s o f t h e f o r m a t i o n o f 1 n m o l o f p r o d u c t p e r m i n u t e . Specific a c t i v -
ity is d e f i n e d in t e r m s o f units p e r m i l l i g r a m o f p r o t e i n ; p r o t e i n is d e t e r -
m i n e d b y t h e m e t h o d o f L o w r y et al. 13 w i t h b o v i n e s e r u m a l b u m i n as
standard.

7.6. The column is washed with the same buffer and eluted with a linear gradient (150 ml
total volume) established between the starting buffer and 1 M triethylamine-carbonate at
the same pH; fractions of 2 ml are collected. PAPS elutes sharply with a peak concentra-
tion in about fraction 38. The major fractions are pooled and brought to dryness at 37° with
a rotary evaporator; the samples are dissolved in water and evaporated again. This proce-
dure is repeated several times to remove traces of triethylamine (yield: 67%). PAPS is
stored in aqueous solution at -20 °.
9 Adapted3with modification from that of Y. Nose and F. Lipmann, J. Biol. Chem. 233, 1348
(1958).
1o Although adenosine 3'-phosphate 5'-phosphosulfate or Y-phosphoadenylylsulfate are the
recommended names for the compound, it was originally designated as 3'-phos-
phoadenosine 5'-phosphosulfate and abbreviated as PAPS. PAPS is retained here as
the abbreviation. A convenient organic synthesis of this nucleotide is in this volume [53].
i1 Because 5% acetone is not inhibitory to the sulfotransferases, this solvent is a useful
medium for stock solutions of the relatively hydrophobic phenols.
12 Sulfotransferase IV is inhibited at high salt concentrations. The amount of enzyme solu-
tion used for its assay, particularly if it is from a salt gradient, should be below 0.25 ml in
order to avoid inhibition.
~30. H. Lowry, N. J. Rosebrough, A. C. Farr, and R. J. Randall, J. Biol. Chem. 193, 265
(1951).
200 ENZYME PREPARATIONS [24]

TABLE I
S U M M A R Y OF T H E P U R I F I C A T I O N OF A R Y L S U L F O T R A N S F E R A S E S I A N D IXa

Specific
Volume Activity Protein activity
Step (ml) (units) (ms) (units/mg)

I. Extract 1390 19.700 43,200 0.46

2. Affi-Gelblue 1140 9,880 1,280 7.7
3. Salt precipitation 57 7,990 650 12
Sulfotransferase I
4A. DEAE-cellulose 59 1,260 41 31
5A. Hydroxylapatite 26 750 5.2 144
7A. Gel filtration 30 600 3.2 187
8A. ATP-agarose 7.3 219 0.88 250
Sulfotransferase II
4B. DEAE-ceUulose 57 1,390 106 15
5B. Hydroxylapatite 22 700 8.6 82
6B. CM-cellulose 7.4 400 2.8 142
7B. Gel filtration 26 347 1.7 199
8B. ATP-agarose 7.3 138 0.49 282

a From Sekura and Jakoby,3 by permission of the Journal of Biological Chemistry.

Purification Procedure
General. Purification o f all four aryl sulfotransferases is conducted
together (Steps 1 through 4) until the stage o f partial separation upon
elution from DEAE/cellulose. The procedure has been applied to lots of
about 50 rat livers (0.5 kg) obtained from well-fed, male Sprague-Dawley TM
rats weighing about 200 g. Livers are maintained at - 8 0 ° prior to use.
Unless otherwise specified, all operations are conducted at 4°. The results
o f purification are summarized in Tables I and II.
Step 1. Extraction. F r o z e n rat livers are allowed to thaw in 1.5 liters of
Buffer A (10 m M Tris-HCl, 0.25 M sucrose, and 3 m M
2-mercaptoethanol, adjusted to p H 7.4 at 25°) and homogenized for two
30-sec periods in a Waring blender maintained in a cold room. The
homogenate is centrifuged at 140,000 g for 90 min and, after removal by
aspiration o f the low density material at the top of the tube, the superna-
tant fluid is collected.
Step 2. Affi-Gel Blue. Without further treatment, the protein solution is
applied to a column (4 x 22 cm) o f Affi-Gel Blue (BioRad, 100-200 mesh)
that has been equilibrated with Buffer A. The column is sequentially

~4The livers were obtained in a frozen state (Dri-Ice) from ARS Sprague-Dawley, Madison,
WI.
[24] Aavc SULFOTRANSFERASES 201

TABLE II
SUMMARY OF PURIFICATION FOR ARYL SULFOTRANSFERASE IV

Specific
Volume Activity Protein activity
Step (ml) (units) (rag) (units/mg)

I. Extract 1430 77,000 41,000 1.9

2. Afli-Gelblue 67 18,300 2,310 7.9
3. DEAE-cellulose 53 4,450 107 107
4. Hydroxylapatite 17 3,640 20.8 175
5. ATP-agarose 12.9 1,740 3.2 540

eluted at a flow rate of about 120 ml per hour with each of the following
solutions: (1) 1.1 liters of Buffer A; (2) a linear gradient between 1.4 liters
of Buffer A and 1.4 liters of Buffer A containing 0.4 M NaCI; (3) a linear
gradient consisting o f 220 ml of Buffer A containing 0.4 M NaCI and an
equal volume of Buffer A containing 2 M NaCI.
Although a significant amount of activity appears in the initial column
washings, the major portion elutes as a broad peak, beginning in the later
part of the first salt gradient; the second, higher salt gradient is necessary
to fully remove the bound enzymes. Activities for aryl sulfotransferases I
and II (assayed at p H 7.4) and III and IV (assayed at p H 5.5) elute in an
entirely parallel pattern. The active fractions are pooled to yield about 1
liter.
Step 3. Salt Precipitation. The sulfotransferases are concentrated by
precipitation upon addition of 313 g per liter of solid ammonium sulfate
during a half-hour period. After stirring for another 30 min, the suspension
is centrifuged at 16,000 g for 30 min and the precipitate is dissolved in 2.5
m M sodium phosphate at pH 6.8 containing 3 m M 2-mercaptoethanol and
0.25 M sucrose. The enzyme is dialyzed overnight against two 4-liter
changes of the same buffer; the precipitate accumulated upon dialysis is
removed by centrifugation.
Step 4. DEAE-Cellulose. The protein solution is adjusted to pH 7.9
with 1 M Tris base and is applied to a column (2.5 × 17 cm) of DEAE-
cellulose (DE-52, Whatman) previously equilibrated with Buffer A. The
column is washed with 500 ml of Buffer A and eluted with a linear gradient
established between 400 ml o f Buffer A and 400 ml of Buffer A that is 0.3
M in sodium chloride.
This procedure allows the elution of aryl sulfotransferases I and II, in
that order, followed by aryl sulfotransferases III and IV as a single peak of
activity. Thus, there is significant separation o f the transferases, except
separation of transferases III and IV from each other. The solution con-
202 ENZYMEPREPARATIONS [24]

taining enzymes III and IV is concentrated about 2-fold with an Amicon

ultrafiltration cell and a PM-10 membrane. Each of the three pools of
enzyme is separately dialyzed overnight against two 2-liter changes of
Buffer B (I0 mM sodium phosphate, 0.25 M sucrose, and 3 mM
2-mercaptoethanol; adjusted to pH 6.8).

Sulfotransferases I and H
Step 5. Hydroxylapatite. The dialyzed protein solutions of transferase I
and transferase II are charged onto separate columns (1.5 x 9.5 cm) of
hydroxylapatite that are equilibrated with Buffer B. The columns are
washed with 30 ml of the same buffer and eluted with a linear gradient
established between 150 ml of Buffer B and 150 ml of Buffer B containing
300 mM potassium phosphate; fractions of about 2.5 ml are collected.
Step 6. Carboxymethyl-Cellulose. Transferase II from Step 5 is dialyzed
overnight against two 2-liter changes of Buffer C (10 mM sodium phos-
phate, 3 mM 2-mercaptoethanol, 0.2 mM Na~EDTA, and 0.25 M sucrose;
adjusted to pH 6.5). The dialyzed material is applied to a column (0.9 x 9
cm) of CM52 (Whatman) that had been equilibrated with Buffer C. The
column is washed with 30 ml of Buffer C and eluted with a linear gradient
established between 75 ml of Buffer C and 75 ml of the same buffer
containing 0.2M NaCI. Fractions of 2 ml are collected at a flow rate of about
40 ml per hour. Contaminating protein appears in the initial column wash-
ings, whereas the enzyme elutes with the gradient as a symmetrical peak
corresponding to the major protein peak. Because of appreciable losses in
activity, without significant gain in purification, it is not recommended that
transferase I be subjected to this step.
Step 7. Gel Filtration. Transferase I from Step 5 and transferase II from
Step 6 are separately concentrated to about 10 ml with an Amicon ultra_fil-
tration cell using a PM-10 membrane. The concentrated materials are
applied to columns (2.0 x 85 cm) of Sephadex G-100 equilibrated with
Buffer D (50 mM sodium phosphate, 0.25 M sucrose, 0.2 mM EDTA, and
3 mM 2-mercaptoethanol; adjusted to pH 6.5). Elution is conducted with
the same buffer at a flow rate of about 20 ml per hour. Each enzyme
appears as a slightly asymmetric protein peak that corresponds closely
with enzyme activity. At this stage, both enzymes represent more than
97% of the protein based on patterns obtained after SDS-gel elec-
trophoresis.
Step 8. ATP-Agarose. Half of the pooled fractions of transferases I and
II are applied, at 25°, to columns (0.5 × 1.5 cm) of agarose-hexane-
adenosine 5'-triphosphate (P-L Biochemicals) that had been equilibrated
with Buffer D. The columns are washed with 10 ml of the buffer and eluted
with a linear gradient established between 20 ml of Buffer D and 20 ml of
[24] ARYL SULFOTRANSFERASES 203

Buffer D containing 45 ttM PAPS. Fractions of 1 ml are collected at a flow

rate of 40 ml per hour. The enzymes are each eluted as single peaks in
which activity is coincident with protein. Despite the high purity of each
of the transferases at earlier stages of preparation as determined by SDS-
gel electrophoresis, this step increases specific activity by more than 30%,
suggesting that the affinity resin may be removing not only traces of con-
taminating protein, but also inactive aryl sulfotransferases.

Aryl Sulfotransferases III and IV

Step 9. Hydroxylapatite. The dialyzed and concentrated solution ob-
tained after DEAE-cellulose chromatography (Step 4) is applied to a col-
umn of hydroxylapatiteTM equilibrated with Buffer B. The column is
washed with 50 ml of Buffer B and eluted with a linear gradient estab-
lished between 250 ml of Buffer B and 250 ml of Buffer E (0.3 M potassium
phosphate, 0.25 M sucrose, and 3 mM 2-mercaptoethanol; adjusted to pH
6.8). Sulfotransferases III and IV are partially resolved on this column;
transferase III elutes as a trailing peak immediately after transferase IV.
Higher ionic strength is necessary to elute transferases I and II, if they are
present.
Step 10. ATP-Agarose. Pooled fractions of sulfotransferase IV from
Step 9 are combined and dialyzed against 2 liters of Buffer F (0.1 M
sodium acetate, 0.25 M sucrose, and 3 mM 2-mercaptoethanol; adjusted
to pH 5.5). The dialyzed sample (5 ml) is applied to a column (0.9 × 2.5
cm) of agarose-hexane-adenosine 5'-triphosphate (P-L Biochemicals)
previously equilibrated with Buffer F at 25°. The column at 25° is washed
with 30 ml of Buffer F and then with 20 ml of Buffer C containing 20/zM
PAPS. Sulfotransferase IV is eluted from the column at the beginning of
the PAPS wash as a slightly trailing peak.
Step 11. Isoelectric Focusing. For studies in which PAPS must be ex-
cluded from the enzyme, isoelectric focusing may be used instead of Step
10. After chromatography on hydroxylapatite, the pooled fractions of
transferase IV are concentrated in an Amicon cell with a PM-10 mem-
brane and then dialyzed against two 2-liter changes of a pH 6.8 buffer
containing 0.25 M sucrose, 2.5 mM sodium phosphate, and 3 mM
2-mercaptoethanol. The resulting protein solution is mixed with a linear
sucrose gradient in a l l0-ml isoelectric focusing column (LKB), using
1.5% (v/v) Ampholines (pH 3.5-10). Electrofocusing is carried out for 16
hr at constant power (15 W). The peak activity of sulfotransferase IV is
collected in fractions with a pH between 5.6 and 5.8. This is well sepa-
rated from the peak of sulfotransferase III activity, which is at pH 6.4.
The enzyme prepared by isoelectric focusing, rather than by Step 10, is
also homogeneous and at the same specific activity.
204 ENZYME PREPARATIONS [24]

Storage
The transferases may be stored at 4° in sodium phosphate at pH 7.0 in
the presence of 0.25 M sucrose, 5 mM mercaptoethanol, and 3 mM
sodium azide; transferases I, II, and IV lose about 5% of their activity per
week at a protein concentration of between 0.5 and 1 mg per milliliter.
Transferase III may deteriorate at - 8 0 ° at any stage of purification and is
definitely unstable to isoelectric focusing.

Properties
Physical Properties. 1,3,4 The three sulfotransferases that have been
studied in detail, I, II, and IV, appear to be homogeneous by the criteria of
disc gel electrophoresis and SDS-gel electrophoresis. Molecular weight,
subunit size, and the isoelectric point have been determined for all four
enzymes (Table III). Amino acid analyses are available for transferases I,
II, and IV. 3"4
pH Optima and Salt Effects. The effects of pH and salt on these en-
zymes are complex and are detailed elsewhere. 3"4The major pH optimum
for 2-naphthol is at 6.5 for transferases I and II and at 5.5 for transferases
III and IV. The two groups of enzymes are also distinguishable on the
basis of their activity at high salt concentrations: transferases I and II are
activated appreciably in the presence of 0.5 M sodium chloride or potas-
sium chloride, whereas transferase IV is strongly inhibited.
The difficulty in evaluating a possible phenolic substrate is that homo-
geneous preparations of these enzymes may display, with some sub-
strates, two pH optima for a single enzyme and have a range of three pH
units within which the pH optimum for a phenol can fall for any one
transferase. For example, at pH 5.5 (the pH optimum for 2-naphthol),

TABLE III
PROPERTIES OF ARYL SULFOTRANSFERASESa

Aryl sulfotransferase

Property I II III IV

Molecular weight b 64,000 64,000 61,000 61,000

Subunit molecular weight C 35,000 35,000 33,500 33,500
Isoelectric point 8.1 6.9 6.4 5.8

a Data from Refs. 9-11.

b Estimated by gel filtration with Sephadex G-100.
c Estimated by sodium dodecyl sulfate disc gel electrophoresis.
[24] ARYLSULFOTRANSFERASES 205

T A B L E IV
KINETIC CONSTANTS FOR THE ARYL SULFOTRANSFERASES a

Aryl sulfotransferase

Ib II b IV ~

Km keat Km keat Km keat

Substrate (raM) (min-') (mM) (rain -1) (raM) (min -~)

2-Naphthol 0.06 47 0.09 54 0.10 42

Phenol 1.8 6.8 2.6 7.3 0.92 1.8
4-Chlorophenol 1.5 48 1.2 40 0.34 18
4-Methoxyphenol 4.2 93 6.5 120 1.4 27
Epinephrine a N N N N 0.43 7.3
Tyrosine methyl ester a N N N N 0.91 14
Tyramine e N N N N 0.46 2.8
2-Cyanoethyl-N-
hydroxythioacetimidate d N N N N 6.9 14

" Apparent K m values were obtained with 0.1 m M PAPS. Data from Refs. 9-11. N indi-
cates that no detectable sulfation occurred.
Data obtained at p H 6.5 unless otherwise indicated.
c Data obtained at pH 5.5 unless otherwise indicated.
d D e t e r m i n e d at p H 7.9.
e Determined at p H 9.0.

transferase IV does not catalyze the formation of sulfate esters (within the
limits of the assay method, 7 from tyrosine methyl ester, epinephrine, oc-
topamine, tyramine, serotonin, and N-acetylserotonin. Yet all are active
at pH 9.0 and some at pH 7.9. Only the last two of these compounds serve
as substrates for transferase I, which has a pH optimum for 2-naphthol at
pH 6.5; however, with this enzyme, serotonin is active between pH 6.5
and 9.0, as is N-acetylserotonin, although the latter has its pH optimum at
pH 6.5. It will be obvious that the pH activity relationship must be deter-
mined for each substrate in which there is an interest.
Substrates. The enzymes are specific for PAPS with apparent Km in the
standard assay system of 6.5 ftM, 12 gM, and 24/zM for transferases I,
II, and IV, respectively.
The activity of these enzymes with simple phenols has been de-
tailed.l'3-~ Aminophenols are better substrates for transferase IV than for
I or II. Specifically, tyrosine methyl ester, epinephrine, and such peptides
that contain N-terminal tyrosine residues (e.g., certain of the enkephalins 4
and cholecystokinin heptapeptide 15) are substrates for transferase IV but
not for I and II. Neither tyrosine itself nor peptides in which tyrosine is in

~5 M. Beinfeld and M. W. Duffel, unpublished data.

206 ENZYME PREPARATIONS [25]

a position other than N-terminal are substrates for any of the purified
sulfotransferases. 4 Transferase IV, but not transferases ! or II, is active
with organic h y d r o x y l a m i n e s such as N-hydroxy-2-acetylaminofluorene
and 2-cyanoethyl-N-hydroxythioacetimidate. 1'4 Kinetic constants for a
few phenols and one h y d r o x y l a m i n e derivative are presented in Table IV.
Detailed studies of the substrate specificity of aryl sulfotransferase IV
have indicated that electron-withdrawing substituents decrease the maxi-
mal velocity o f phenol sulfation. 16 Phenols with m a n y strongly electron-
withdrawing substituents (e.g., pentachlorophenol) are dead-end inhib-
itors. 16
Inhibitors. With phenols showing a low Km, there is generally pro-
nounced substrate inhibition at high substrate concentrations. The en-
z y m e s are also inhibited b y thiol reagents, 17 reaction products (i.e.,
adenosine 3 ' , 5 ' - b i s p h o s p h a t e or aryl sulfates) and dead-end inhibitors
such as 2,6-dichloro-4-nitrophenol and pentachlorophenol, t8
i0 M. W. Duffel and W. B. Jakoby, J. Biol. Chem., in press.
1~D. J. Barford and J. G. Jones, Biochem. J. 123, 427 (1971).
is G. J. Mulder and E. Scholtens, Biochem. J. 165, 553 (1977).

[25] Hydroxysteroid Sulfotransferase

By ELLEN SUE LYON, CAROL J. MARCUS, JUN-LAN WANG, and
WILLIAM B. JAKOBY

ROH + adenosine T-phosphate 5'-phosphosulfate -~

ROSO3H + adenosine 3',5'-bisphosphate
The h o m o g e n e o u s preparations of h y d r o x y s t e r o i d sulfotransferase
(EC 2.8.2.2) f r o m rat liver catalyze the formation o f sulfate monoesters
from PAPS 1 with a v e r y large n u m b e r and variety o f p r i m a r y and second-
ary alcohols that include a m o n g t h e m p r i m a r y and secondary h y d r o x y -
steroids.

Assay M e t h o d
Principle. T h e assays for hydroxystcroid sulfotransfcrase arc based on
the separation of radiolabeled substrate from product on the basis of
polarity. A filter method, 2 using radiolabeled steroid as substrate, is sim-
i Although adenosine Y-phosphate Y-phosphosulfate or 3'-phosphoadenylylsulfate are the
recommended names for the compound, it was originally designated as 3'-
phosphoadcnosine 5'-phosphosulfate and abbreviated as PAPS. This abbreviation is re-
tained here.
2 R. D. Sekura, C. J. Marcus, E. S. Lyon, and W. B. Jakoby, Anal. Biochem. 95, 82 (1979).

Copyright © 1981 by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. 77 All rights of reproduction in any form reserved.
ISBN 0-12-181977-9